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The effectiveness of oral HIV preexposure prophylaxis (PrEP) strongly depends on maintaining adherence. We investigated the association between substance use and PrEP adherence, as well as incident sexually transmitted infections (STIs) in a high-risk cohort of 394 participants (391 men who have sex with men and 3 transgender women) who were enrolled in a PrEP demonstration project. We assessed baseline and ongoing substance use over a 48-week period for stimulants and nonstimulant substances and for each substance separately. We measured PrEP adherence by using dried blood spots to obtain levels of tenofovir diphosphate. No differences in these levels were found between substance users and nonsubstance users. Baseline stimulant use was strongly associated (odds ratio 3.4; p<0.001) with incident STIs during the study. Thus, PrEP adherence was not decreased by substance use. Because substance users had increased rates of STIs, indicating higher-risk behavior, they might be excellent candidates for PrEP.